Tobacco smoke regulates the expression and ... - The FASEB Journal

The FASEB Journal • Research Communication

Tobacco smoke regulates the expression and activity of microsomal prostaglandin E synthase-1: role of prostacyclin and NADPH-oxidase Silvia S. Barbieri,*,1 Patrizia Amadio,† Sara Gianellini,† Elena Zacchi,† Babette B. Weksler,‡ and Elena Tremoli*,† *Centro Cardiologico Monzino, Istituto di Ricovero e Cura a Carattere Scientifico, Milan, Italy; † Department of Pharmacological Sciences, University of Milan, Milan, Italy; ‡Division of Hematology–Medical Oncology, Weill Medical College of Cornell University, New York, New York, USA Tobacco smoke (TS) interacts with interleukin-1␤ (IL-1␤) to modulate generation of reactive oxygen species (ROS) and expression of cyclooxygenase-2. We explored molecular mechanisms by which TS/ IL-1␤ alters expression and activity of microsomal-prostaglandin E synthase-1 (mPGES-1) and of prostacyclin synthase (PGIS) in mouse cardiac endothelial cells. TS (EC50 ⬃5 puffs/L) interacting with IL-1␤ (2 ␮g/L) upregulates PGE2 production and mPGES-1 expression, reaching a plateau at 4 – 6 h, but down-regulates prostacyclin (PGI2) release by increasing IL-1␤-mediated PGIS tyrosine nitration. Inhibition of NADPH-oxidase, achieved pharmacologically and/or by silencing its catalytic subunit p47phox, or exogenous PGI2 (carbaprostacyclin; IC50 ⬃5 ␮M) prevents production of both ROS and PGE2, and negatively modulates mPGES-1 expression induced by TS/IL-1␤. Moreover, inhibiting PGI2, either using PGIS siRNA and/or CAY10441 (EC50 ⬃20 nM), a PGI2 receptor antagonist, increases NADPH-oxidase activation, mPGES-1 synthesis, and PGE2 production. Finally, lower PGI2 levels associated with higher PGIS tyrosine nitration, p47phox translocation to the membrane (an index of activation of NADPH-oxidase), and mPGES-1 expression and activity were detected in cardiovascular tissues of ApoEⴚ/ⴚ mice exposed to cigarette smoke compared to control mice. In conclusion, cigarette smoke in association with cytokines alters the balance between PGI2/PGE2, reducing PGI2 production and increasing synthesis and activity of mPGES-1 via NADPH-oxidase activation, predisposing to development of pathological conditions.—Barbieri, S. S., Amadio, P., Gianellini, S., Zacchi, E., Weksler, B. B., Tremoli, E. Tobacco smoke regulates the expression and activity of microsomal prostaglandin E synthase-1: role of prostacyclin and NADPHoxidase. FASEB J. 25, 3731–3740 (2011). www.fasebj.org ABSTRACT

Key Words: prostanoids 䡠 reactive oxygen species 䡠 endothelial cells 䡠 signal transduction

Epidemiological and experimental studies show that cigarette smoking increases the incidence of myocardial infarction, coronary heart disease, and progres0892-6638/11/0025-3731 © FASEB

sion of atherosclerosis in humans and in animal models (1– 4). The ability of cigarette smoke to induce endothelial dysfunction and activation of vascular inflammatory genes (1) is a necessary step in the cascade of events that culminates in the erosion of the fibrous cap of the atherosclerotic plaque leading to its subsequent rupture. Prostanoids, such as prostaglandin E2 (PGE2) and prostacyclin (PGI2), are inflammation-modulating lipids generated by endothelium from sequential activation of cyclooxygenases (COX-1 and COX-2) and different specific isomerases, prostaglandin E synthases (PGESs), and prostacyclin synthase (PGIS) that direct specific prostanoid production. Strong evidence links smoking with altered production of prostanoids (PGE2 and PGI2; refs. 2, 5, 6), and links altered levels of PGI2 or PGE2 with cardiovascular disease (7). In particular, low levels of PGIS are detected in atherosclerotic aortic lesions (8); conversely, inducible microsomal prostaglandin E synthase-1 (mPGES-1) appears to be overexpressed in unstable atherosclerotic plaques (9). These data have also been confirmed using mPGES-1 (10) or IP receptor-knockout mice (11), suggesting a key roles for these genes in atherothrombosis. Oxidative stress strongly contributes to the pathophysiology of vascular diseases (12), and its level may be regulated by cigarette smoke (13). NADPH-oxidase, the major enzyme involved in producing superoxide and reactive oxygen species (ROS) in endothelial cells, consists of a membrane-localized cytochrome b558 composed of Nox and p22phox, and several cytosolic components, including p40phox, p47phox, p67phox, and Rac. Exposure of cells to a variety of stimuli induces the translocation of cytosolic components of NADPHoxidase to the plasma membrane, leading to superoxide production (14). Recent evidence indicates that 1 Correspondence: Centro Cardiologico Monzino, IRCCS, Via Parea 4, 20138 Milano, Italy. E-mail: silviabarbieri@ yahoo.com doi: 10.1096/fj.11-181776 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

3731

cigarette smoke induces formation of this multimeric protein complex with consequent activation of NADPHoxidase and ROS generation (15–17). We investigated whether tobacco smoke (TS) interacting with inflammatory cytokine interleukin-1␤ (IL1␤) alters the levels or activities of mPGES-1 and PGIS in a NADPH-oxidase-dependent manner. In particular, we decided to study the effect of different concentrations of TS (3.2–12.8 puffs/L), all easily achieved in the bloodstream of smokers, in association with the inflammatory cytokine IL-1␤. This cytokine markedly increases in human and animal models of smoking (2, 17, 18), and it plays a key role in the pathogenesis of inflammatory disorders. Our data indicate that exposure of endothelial cells to TS increases the tyrosine nitration of PGIS and decreases PGI2 production induced by IL-1␤. In contrast, TS interacts with IL-1␤ to up-regulate expression and activity of mPGES-1. This dual effect leads to a decrease in vasoprotective PGI2 and an increase in inflammatory PGE2. In particular, we provide evidence that the TS-mediated down-regulation of PGI2 induces the translocation of the p47phox subunit of NADPH-oxidase to the plasma membrane and increases ROS generation, with consequent upregulation of mPGES-1 expression and PGE2 production, suggesting for the first time a direct link of smoking to a simultaneous dual modulation of PGI2 and PGE2 that has proatherogenic consequences.

MATERIALS AND METHODS Cell culture Mouse cardiac endothelial cells (MCECs) were isolated, immortalized, and characterized as described previously (2), and cultured in DMEM with 10 mM penicillin/streptomycin (Life Technologies, Invitrogen, Carlsbad, CA, USA), 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA) and 5% FBS in 0.2% gelatin-coated plates. Cells at confluence were starved for 24 h in fresh DMEM containing 0.5% FBS, then incubated in serum-free medium with or without TS and/or IL-1␤. Preparation of TS extract 2R4F research cigarettes were smoked one at a time in a Borgwaldt piston-controlled apparatus (model RG-1; Borgwaldt, Hamburg, Germany) using the U.S. Federal Trade Commission standard protocol that mimics a standardized human smoking pattern (puff duration, 2 s; frequency, 1 puff/min; volume, 35 ml/puff). The smoke was drawn under sterile conditions through premeasured amounts of sterile PBS (pH 7.4). Smoke dissolved in PBS represents a hydrophilic fraction of mainstream cigarette smoke. Many hydrophobic and volatile mainstream smoke chemicals are indeed eliminated by this procedure. TS dissolved in PBS was quantitated as puffs per liter of PBS, with 1 cigarette yielding ⬃8 puffs drawn into a 10-ml volume. The final concentration of TS in the cell culture medium is expressed as puffs per liter of medium. Aliquots of TS were snap-frozen immediately after preparation and kept in liquid nitrogen until use. Preliminary studies showed that the biological properties of TS were stable under these conditions (19). 3732

Vol. 25

October 2011

Cell incubation Two different clones of MCECs were used with similar results. Cells at confluence were starved 24 h in fresh medium containing 0.5% FBS, then incubated in DMEM 0.5% FBS with or without TS and/or IL-1␤ (Sigma-Aldrich) as indicated. Apocynin, DPI (Sigma-Aldrich), carbaprostacyclin, and CAY10441 (Cayman Chemical, Ann Arbor, MI, USA) were added 2 h before cell stimulation. Prostaglandin assays Prostanoids were determined in MCECs that were exposed to stimuli for 6 h, and prostanoids produced from endogenous arachidonate were measured in culture supernatants. The cells were then washed in PBS and incubated for 30 min with exogenous arachidonic acid (AA; 10 ␮M), and supernatants were collected for measurement of prostanoids produced from exogenous arachidonate. Data are expressed as the difference in prostanoids between normal samples and controls preexposed to indomethacin (10 ␮M), to eliminate possible nonspecific immunoreactivity. Mouse tissue was weighed, homogenized in 0.1 M phosphate buffer (pH 7.4), 1 mM EDTA, and 10 ␮M indomethacin, and centrifuged for 10 min at 12,000 rpm at 4°C, and supernatant was used to detect prostanoid tissue levels (20). 6-Keto prostaglandin F1␣ (6-keto-PGF1␣) and PGE2 levels were measured using PGE2 and 6-keto-PGF-1␣ enzyme immunoassay (EIA) kits (Cayman Chemical). Protein preparation MCECs were washed twice in PBS and lysed in cold RIPA buffer (62.5 mM Tris HCl; 100 mM NaCl; 1% Nonidet P-40; 0.1% Tween 20; 1 mM Na3VO4; 1 mM PMSF; 10 mM Na-pyrophosphate; 10 mM NaF, pH 8.0; and protease inhibitor cocktail). After sonication and centrifugation to remove cell debris, protein yields were quantified using the BCA protein assay kit (Pierce, Thermo Scientific, Waltham, MA, USA). Samples (25 ␮g) were prepared by the Laemmli method, and equivalent amounts of protein were separated by electrophoresis in 7–15% SDS-PAGE gels; specific bands were developed using anti-mPGES-1 or PGIS antibody (Cayman Chemical). Immunoprecipitation Tissues and MCECs were lysed in RIPA buffer for 30 min on ice. The antibody against PGIS (1 ␮g; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to protein A-Sepharose beads (20 ␮l; Sigma-Aldrich) and incubated with gentle rocking overnight at 4°C. Protein A-Sepharose beads were sedimented by brief centrifugation, washed with immunoprecipitation buffer, and suspended in 50 ␮l of immunoprecipitation buffer per sample. Cell lysates (300 ␮g) were incubated with beads for 2 h at 4°C. Subsequently, beads were washed, and the pellet was lysed in 20 ␮l of 4⫻ Laemmli buffer (19). Aliquots (10 ␮l) of immunoprecipitated protein were separated on 7% SDS-PAGE gel and immunoblotted with antibodies to PGIS (Santa Cruz Biotechnology) or to nitrotyrosine (3-NT; Upstate Biotechnology, Waltham, MA, USA). Separation of cytosol/membrane MCECs or mouse tissues were suspended in relaxation buffer (10 mM HEPES, pH 7.6; 3 mM NaCl; 100 mM KCl; 3.5 mM MgCl2; 0.1 mM DTT; 0.1 mM EDTA; 0.5 mM PMSF; and

The FASEB Journal 䡠 www.fasebj.org

BARBIERI ET AL.

protease inhibitor cocktail), sonicated on ice (2⫻10 s) and centrifuged (600 g for 10 min at 4°C) to remove nuclei and unbroken cells. The supernatant was then ultracentrifuged (105 g for 30 min at 4°C). Membranes were washed in relaxation buffer and dissolved in Laemmli sample buffer (21). Samples (30 ␮g) were prepared with the Laemmli method, and equivalent amounts of protein were separated on 10% SDS-PAGE gel and revealed using anti-p47phox antibody (Santa Cruz Biotechnology). Western blot analysis Cells were harvested in appropriate lysis buffer, and blotting was performed as described previously (2). The equivalency of protein loading was confirmed by Ponceau Red staining. Membranes were incubated overnight at 4°C with primary antibodies directed against mPGES-1 (1:250; Cayman Chemical), 3-NT (1:250; Upstate Biotechnology), PGIS, (1:1,000; Santa Cruz Biotechnology) or p47phox (1:500, Santa Cruz Biotechnology), ␤-actin, and tubulin (1:10,000; Sigma-Aldrich). Subsequently, membranes were incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence (EuroClone, Milan, Italy). RNA extraction and RT-PCR Total cellular RNA was isolated from cells with TRIzol reagent (Sigma-Aldrich), and 1.5-␮g aliquots of RNA were reverse transcribed in 20 ␮l of reaction volume using SuperScript II RNase H (Invitrogen). Samples were amplified for 10 min at 25°C, 20 min at 42°C, 5 min at 99°C, and 5 min at 5°C. cDNA (2.5 ␮l) was subjected to 26 cycles of PCR: denaturation at 95°C for 40 s (mPGES-1, ␤-actin, PGIS); annealing at 63°C for 30 s (mPGES-1 and ␤-actin) or 15 s (PGIS); extension at 72°C for 45 s (mPGES-1 and ␤-actin) or 60°C for 1 min (PGIS) in a reaction mixture (25 ␮l) containing 2.5 U TaqDNA polymerase (Invitrogen) and 5 ␮M forward primer and reverse primer. mPGES primers were 5⬘-GAGCCCACCGCAACGACATG-3⬘ and 5⬘-CAGATGGTGGGCCACCTCCC-3⬘; PGIS primers were 5⬘-TCACCACGCACCCATGAG-3⬘, and 5⬘TGGCGGAAGGTATGGAAAACATC-3⬘; ␤-actin primers were 5⬘-GGTCACCCACACTGTGCCCAT-3⬘ and 5⬘-GGATGCCACAGGACTCCATGC-3⬘. All reactions were performed in a Swift Maxi thermocycler (ESCO Life Sciences, Breukelen, Netherlands). PCR products were then separated on agarose gel (1.5% for mPGES-1, 1% for ␤-actin and for PGIS) containing 0.5 mg/L of ethidium bromide and photographed under ultraviolet light. The identity of each PCR product was confirmed by DNA sequencing. Transfection of siRNA MCECs, plated in antibiotic-free medium containing 5% FBS, were grown to 50 –70% confluence. Transfection with siRNAs was performed using transfection reagent and medium (Santa Cruz Biotechnology) to result in a final p47phox siRNA, PGIS siRNA, or control siRNA (all from Santa Cruz Biotechnology) concentration of 50 nM. After 7 h incubation, an equal volume of DMEM/5% FBS was added without removing the transfection mixture. The following day, the cells were retransfected according to the same protocol. After 18 h, the transfected cells were washed and starved for 24 h in DMEM/0.5% FBS. SMOKE REGULATES mPGES-1 VIA PROSTACYCLIN/ROS PATHWAYS

Measurement of ROS production MCECs were loaded with 10 ␮M of 5-(and-6)-chloromethyl-2⬘,7⬘dichlorodihydrofluorescein diacetate acetyl ester (DCFH-DA, a ROS-reacting fluorescent dye) in DMEM without phenol red at 37°C for 1 h. After the incubation, cells were washed in PBS with Ca/Mg, and oxidative activity was assessed. The production of ROS was measured by the intensity of DCF emission at 525 nm (excitation 503 nm; Perkin-Elmer LS 50B) in stained and unstained cells. Data are expressed as the difference in fluorescence (in arbitrary units, AU) calculated as AU ⫽ (It5 ⫺ It0), where I represents the intensity of fluorescence at the specified time points (19). Data were obtained from 4 independent experiments, each performed in triplicate. Animal studies and in vivo experiments All animal experimental procedures were approved by the Weill Medical College Institutional Animal Care and Use Committee. ApoE⫺/⫺ mice aged 9 wk (5 males/group; Jackson Laboratories, Bar Harbor, ME, USA) were fed with standard chow. Mice were secured in fitted polycarbonate chambers and placed into a 12-port nose-only exposure chamber (CH Technologies, Westwood, NJ, USA) for direct inhalation of cigarette smoke. The cigarettes used in this study were 2R4F research cigarettes (Kentucky Tobacco Research Institute, University of Kentucky, Lexington, KY, USA) smoked at the rate of 1 puff/min, 2 s/puff. Puff volume was 35 ml. Mice were treated with either room air or TS for 1 h/d, 7 d/wk for 15 d at a total suspended particulate (TSP) dose of ⬃200 mg/m3/d. At the end of the entire experimental period, animals were deeply anesthetized with isoflurane. After perfusion with saline, the hearts and aortas were isolated en bloc and lysed in a RIPA buffer or processed for cytosol/membrane separation. Data analysis Data are expressed as means ⫾ se are based on ⱖ3 independent experiments. Groups were compared by Student’s test or ANOVA with the Fisher’s LSD posttest when appropriate. Values of P ⬍ 0.05 were considered significant.

RESULTS TS and IL-1␤ interact to modulate PGE2 and PGI2 production In a previous study, we demonstrated that TS interacts with IL-1␤ to up-regulate COX-2 expression in mouse cardiac endothelial cells (2). In the present study, we measured both PGE2 and PGI2 in supernatants of MCEC monolayers that were treated for 6 h with different concentrations of TS (3.2–12.8 puffs/L), IL-1␤ (2 ␮g/L), or TS plus IL-1␤ (TS/IL-1␤); PGI2 was measured as its stable metabolite, 6-keto-PGF1␣. MCECs incubated with TS significantly reduced 6-ketoPGF1␣ levels in the presence of exogenous arachidonate only at the highest concentration of TS used (12.8 puffs/L; Fig. 1A); in contrast, the treatment of MCECs with IL-1␤ (2 ␮g/L) increased the 6-keto-PGF1␣ production 2-fold. However, MCECs treated with the combination of TS/IL-1␤ produced significantly less 6-ketoPGF1␣ (Fig. 1A) and more PGE2 compared to the 3733

Figure 1. Production of 6-keto-PGF1␣ (A) and PGE2 (B) in MCECs exposed to different stimuli. MCECs were incubated with different stimuli for 6 h. Medium was then collected and replaced with fresh medium containing arachidonic acid (10 ␮M); incubation was continued for 30 min. Data, expressed as the difference in prostanoids between samples and control exposed to indomethacin (10 ␮M), represent means ⫾ se of 6 independent experiments, each performed in duplicate. *P ⬍ 0.05, **P ⬍ 0.01 vs. control; #P ⬍ 0.05 vs. TS; °°P ⬍ 0.01 vs. IL-1␤.

control cells stimulated with either TS (12.8 puffs/L) or IL-1␤ alone (Fig. 1B). MCECs exposed to the highest concentrations of TS combined with IL-1␤ further increased PGE2 production (Fig. 1B). Similar results were obtained from cells preincubated with endogenous arachidonate (data not shown), suggesting that no inactivation of prostanoids resulted from exposure to TS. Additional evidence that TS and/or IL-1␤ did not interact directly with prostanoids is also provided by the fact that the addition of exogenous PGI2 or PGE2 (1␮M) to MCECs treated with indomethacin (10 ␮M) resulted in no decrease in the amount of these added prostanoids after 6 h of incubation with TS and/or IL-1␤ (data not shown). We then examined mRNA and protein expression of the relevant terminal prostaglandin synthases, namely PGIS and mPGES-1. No change in either PGIS mRNA or protein was detected in our experimental conditions (Fig. 2A–C). Since peroxynitrite (ONOO⫺) has been described selectively to inhibit PGIS activity (22), we determined whether the lower PGI2 release following MCEC exposure to TS/IL-1␤ was caused by increased tyrosine nitration of PGIS. After 30 min of TS/IL-1␤ exposure, tyrosine nitration of PGIS was significantly increased (Fig. 2D). We additionally observed that, by itself, only the highest concentration of TS (12.8 puffs/L) up-regulated mPGES-1 protein, an effect seen after 6 h of exposure of MCECs to TS (Fig. 2B, C). However, treatment of MCECs with TS 6.4 or 12.8 puffs/L together with IL-1␤ increased mPGES-1 mRNA and protein compared either to control unstimulated cells or cells exposed to each single stimulus alone (Fig. 2A, B). Moreover, the combination of TS (6.4 puffs/L) and IL-1␤ (TS/IL-1␤) induced the expression of mPGES-1 in a time-dependent manner, reaching a plateau at 4 – 6 h (Fig. 2C). 3734

Vol. 25

October 2011

Taken together, these data suggest that exposure of MCEC to TS/IL-1␤ inhibits activity of PGIS, whereas it increases synthesis and activation of mPGES-1. PGI2 modulates expression and activity of mPGES-1 induced by TS/IL-1␤ To assess whether PGI2 itself modulates mPGES-1, we pretreated TS/IL-1␤-exposed MCECs with carbaprostacyclin (2.5–10 ␮M), a stable PGI2 analog. As shown in Fig. 3A, carbaprostacyclin counteracted, in a dose-dependent manner, the effect of TS/IL-1␤ on mPGES-1 expression. We then confirmed the effect of PGI2, or its analogs, on mPGES-1 by transfecting the MCECs with PGIS-specific siRNA, or by incubating the cells with CAY10441 (10 – 40 nM), an antagonist of the PGI2 receptor (IP). Inhibition of the PGI2/IP receptor pathway with CAY10441 or with PGIS-specific siRNA significantly increased mPGES-1 expression (Fig. 3B, C) and/or activity (Fig. 3D) induced by IL-1␤. Surprisingly and unexpectedly, the reduction of basal levels of endogenous PGI2, mediated by PGIS-specific siRNA or treatment with CAY10441, slightly regulated the expression and activity of mPGES-1 compared to cells transfected with nonspecific siRNA or to control cells, respectively (Fig. 3B–D). Collectively, these results indicate that, in our experimental conditions, the activation of mPGES-1 is negatively regulated by PGI2. PGI2 decreased the activation of NADPH-oxidase in MCEC treated with TS/IL-1␤ It has been recently shown that TS cooperates with IL-1␤ to increase production of ROS (2), and that PGI2 attenuates NADPH-oxidase activity (23). We examined here whether TS/IL-1␤ exposure increases the translo-


BARBIERI ET AL.

Figure 2. TS plus IL-1␤ modulates expression of mPGES-1 and tyrosine nitration of PGIS. A) Total cellular mRNA was extracted after 1 h of exposure to TS (6.4 puffs/L) and/or IL-1␤ (2 ␮g/L), and PGIS and mPGES-1 were analyzed by RT-PCR. B, C) Concentrationdependent (B) and time-dependent (C) effects of TS and/or IL-1␤ on PGIS and mPGES-1 expression by Western blot analysis. D) Extracts of cells stimulated for 30 min were immunoprecipitated with anti-PGIS (IP: PGIS) and immunoblotted with anti-nitrotyrosine (3-NT). Each panel is representative of 3 independent experiments.

cation of p47phox from cytoplasm to the plasma membrane of MCECs, as a marker of the in vitro activation of NADPH-oxidase, and therefore, whether the PGI2 pathway modulates NADPH-oxidase activity in our experimental conditions. p47phox in unstimulated cells or in cells treated with TS was mainly localized in the cytosolic fraction. However, exposure to IL-1␤ induced p47phox subunit redistribution from cytosol to plasma membrane. Moreover, the incubation with TS/IL-1␤ led to the complete translocation of p47phox subunit to the plasma membrane (Fig. 4A, Supplemental Fig. S1A). Incubation with DPI (5 ␮M) or apocynin (15 ␮M) totally prevented ROS production induced by TS/IL-1␤ (data

not shown), indicating that NADPH-oxidase plays a key role in ROS generation in this setting. To assess whether the activation of a PGI2-dependent pathway modulates the production of NADPH-oxidaseinduced ROS in our experimental conditions, we treated MCECs with IL-1␤ and/or TS in the presence of carbaprostacyclin or CAY10441. Both p47phox translocation and ROS production induced by TS/IL-1␤ were almost completely abolished in the presence of carbaprostacyclin (Fig. 4B, D and Supplemental Fig. S1B). In contrast, in MCECs treated with CAY10441 with or without IL-1␤, NADPH-oxidase activity increased dramatically compared to control or IL-1␤, respectively

Figure 3. Role of PGI2 in the TS/IL-1␤-induced up-regulation of mPGES-1 in endothelial cells. A, B) Confluent MCECs were pretreated with different concentrations of carbaprostacyclin (2.5–10 ␮M) for 2 h and then stimulated by TS/IL-1␤ (A) or exposed to different concentration of CAY10441 (10 – 40 nM) with or without IL-1␤ for 6 h (B). C) MCECs were transfected with PGIS siRNA or nonspecific siRNA and then stimulated for 6 h with TS and/or IL-1␤. Cell lysates were processed by Western blot analysis to detect mPGES-1. Each panel is representative of 4 independent experiments. D) Medium was then collected and replaced with new medium containing arachidonic acid (10 ␮M); incubation was continued for 30 min. Production of PGI2 and PGE2 in MCECs transfected with PGIS siRNA or nonspecific siRNA was measured by EIA. Data are expressed as means ⫾ se of 4 independent experiments. *P ⬍ 0.05, **P ⬍ 0.01 vs. control; #P ⬍ 0.05 vs. IL-1␤.

SMOKE REGULATES mPGES-1 VIA PROSTACYCLIN/ROS PATHWAYS

3735

Figure 4. Effect of PGI2 on NADPH oxidase activation mediated by TS/IL-1␤. A–C) p47phox translocation from cytoplasm to the plasma membrane after stimulation with TS and/or IL-1␤ (A), TS/IL-1␤ with or without carbaprostacyclin (10 ␮M; B), or IL-1␤ and/or CAY10441 (40 nM; C), by Western blot analysis. Each panel is representative of 3 independent experiments. D–F) MCECs were pretreated with or without carbaprostacyclin (10 ␮M; D) or CAY10441 (40 nM; E), or transfected with nonspecific siRNA or PGIS siRNA (F), and then stimulated with TS and/or IL-1␤. Intracellular ROS production was detected using DCFH-DA method after 15 min incubation. Data are expressed as means ⫾ se. *P ⬍ 0.05, **P ⬍ 0.01 vs. control; #P ⬍ 0.05, ##P ⬍ 0.01 vs. IL-1␤; °P ⬍ 0.05 vs. CAY10441; $$P ⬍ 0.01 vs. PGIS siRNA control.

(Fig. 4C, E and Supplemental Fig. S1C). In addition, the role of PGI2 in raising ROS production was further confirmed in experiments carried out using PGISspecific siRNA (Fig. 4F). In our experimental conditions, ROS production is not modified by treatment with PGE2 (0.5 ␮M) or PGE2 plus TS/IL-1␤ compared to ROS production by control or by cells treated with TS/IL-1␤, respectively (Supplemental Fig. S1D). In addition, the treatment of MCECs with CAY10526, the inhibitor of mPGES-1 activity, had no effect on ROS production induced by TS/IL-1␤, suggesting that the PGE2 is not involved in the early modulation of TS- and/or IL-1␤-mediated ROS production (Supplemental Fig. S1D). Overall, these data suggest that a decrease in TS/IL1␤-mediated PGI2 induces ROS generation, most likely via an increase in NADPH-oxidase activity. mPGES-1 expression and activity are mediated by NADPH oxidase activation that is induced by TS/IL-1␤ To explore further whether NADPH-oxidase modulates mPGES-1 expression and activity, we preincubated 3736

Vol. 25

October 2011

MCECs either with pharmacological inhibitors of NADPH-oxidase (apocynin or DPI) or p47phox-specific siRNA before exposing the cells to TS/IL-1␤. As shown, apocynin, DPI, and p47phox-specific siRNA treatment of MCECs decreased the effect of TS/IL-1␤ on mPGES-1 protein (Fig. 5A, B), and PGE2 production (Fig. 5C). Thus, ROS produced by TS/IL-1␤-mediated activation of NADPH-oxidase are responsible for the observed increase in mPGES-1 expression and activity. Cigarette smoke exposure increased tyrosine nitration of PGIS, p47phox translocation, and mPGES-1 expression and activity and reduced PGI2 production in ApoE-knockout mice We extended our in vitro observations by testing the effect of smoking exposure on prostanoid production, tyrosine nitration of PGIS, p47phox translocation, and mPGES-1 expression in cardiovascular tissue of ApoEknockout mice, using the same animal protocol that we have used in the previous study. Indeed, the exposure


BARBIERI ET AL.

Figure 5. Role of NADPH-oxidase on TS/IL1␤-induced mPGES-1 expression and activity. MCECs were preincubated for 2 h with or without apocynin (15 ␮M) or DPI (5 ␮M) (A) or transfected with p47phox siRNA or with nonspecific siRNA (B, C), then stimulated for 6 h with TS/IL-1␤. A, B) Lysates of endothelial cells were processed for mPGES-1 (A, B) and p47phox (B) in Western blot analysis. Each panel is representative of 3 independent experiments. C) Culture supernatant was tested for released PGE2 by EIA. Data are expressed as means ⫾ se of 4 independent experiments, each performed in duplicate. *P ⬍ 0.01.

of mice to cigarette smoke for 1 h/d for 15 d was sufficient to increase IL-1␤ serum levels and COX-2 expression in cardiovascular tissue of smoke-exposed mice (2). In accord with our previous observations obtained in isolated MCECs, we observed that cigarette smoke exposure significantly modified the tissue levels of prostanoids, reducing 6-keto-PGF1␣ production and increasing PGE2 generation compared to sham-treated mice (P⬍0.05; Fig. 6A). Similarly, we observed a significant induction of tyrosine nitration of PGIS (3NTPGIS; 260%; P⬍0.05; Fig. 6B), an enhancement of p47phox translocation from the cytosol to the plasma membrane (316%; P⬍0.01; Fig. 6C), and an increase in

mPGES-1 (213%; P⬍0.01; Fig. 6D). Taken together, these data suggest that cigarette smoking modulates the 3NT-PGIS/NADPH-oxidase/mPGES-1 pathway in cardiovascular tissue in vivo as well as in vitro.

DISCUSSION In this study, we explored the mechanisms by which cigarette smoke modulates the production of two major prostanoids, PGE2 and PGI2, both of which have key roles in endothelial dysfunction and in vascular remodeling that occur in atherosclerosis. We show here that cigarette smoke cooperates with

Figure 6. Effect of cigarette smoke exposure on 6-keto-PGF1␣ and PGE2, nitration of PGIS, p47phox translocation, and mPGES-1 expression in ApoE-knockout mice. A) 6-Keto-PGF1␣ and PGE2 from tissue were measured by EIA Data are expressed as means ⫾ se. B, C) Tyrosine nitration of PGIS was analyzed by immunoprecipitation with anti-PGIS (IP: PGIS) and immunoblotting with anti-nitrotyrosine (3-NT) antibody (B) and membrane translocation of p47phox (C). D) mPGES-1 expression was detected in cardiovascular tissue by Western blot analysis. Changes in protein content are shown. *P ⬍ 0.05, **P ⬍ 0.01; n ⫽ 5/group. SMOKE REGULATES mPGES-1 VIA PROSTACYCLIN/ROS PATHWAYS

3737

inflammatory cytokines, such as IL-1␤, to inhibit the activity of PGIS; to induce the activation of NADPHoxidase via inhibition of PGI2 production; and to increase the expression of PGES and the production of PGE2 by a PGI2/p47phox-dependent pathway. As recently shown, plasma and tissue levels of IL-1␤ increase markedly in smokers and play an important role in vascular dysfunction and cardiovascular disease (2, 24 –27). In fact, in vivo exposure to TS induces pentraxin-3 (PTX3) expression in lung pulmonary endothelial cells in an IL-1-dependent manner (27). We postulate that the lung, leukocytes, and platelets are the likely sites of origin of these cytokines. The inflammatory events that develop in lung tissue following the activation of leukocytes lead to increased release of inflammatory cytokines into the bloodstream, an event that, in turn, results in endothelial dysfunction. Indeed, it has been shown that exposure to cigarette smoke increases the secretion of IL-1␤ in lavage fluid (18), as well as in mononuclear blood cells of smokers compared with nonsmokers (28). Moreover, vascular expression of IL-1␤ mRNA is significantly increased both by smoking and by exposure to TS extract (17), suggesting that TS extract by itself can regulate the expression IL-1␤. It is pertinent that deletion of IL-1 receptors reduces emphysema in mice exposed to cigarette smoke (25). Conversely, the IL-1␤-511T allelic polymorphism, which increases the production of IL-1␤, is associated with atherosclerosis and is now considered a risk factor for this disease (29). Precisely for this reason, we have concentrated on the effects of whole-TS interaction with IL-1␤ as the major focus of the work, because we can assume that the results that we obtained reflect what happens in the vascular endothelium of persons who are exposed to cigarette smoke. We also show for the first time, both in vitro and in vivo, that exposure to cigarette smoke increases the expression and/or the activity of mPGES-1, and we confirm recently published data that cigarette smoke reduces the production of PGI2 without inducing alterations in expression of PGIS protein (30). Of interest, we know from previous studies that TS cooperates with other cytokines such as TNF-␣ IL-1␤ to increase COX-2 expression and PGE2 production (2). In particular, we showed that both TS (12.8 puffs/L) and TS (6.4 puffs/L)/IL-1␤ increased COX-2 mRNA and protein levels (2). We now confirm that TS also cooperates with thrombin not only to up-regulate the levels of mPGES-1 and PGE2 but also to decrease PGI2 production (Supplemental Fig. S2). Vascular COX-2 represents one important rate-limiting step for prostanoid production. As a key corollary, we provide evidence that cotreatment with TS/IL-1␤ increases the tyrosine nitration of PGIS, an effect that inactivates PGIS by nitration of tyrosine 430 at the active site (31) through a reaction catalyzed by the ferric iron of this heme-thiolate (P450) protein (32), with consequent blockade of PGI2 production. We show that in our models, both increased 3738

Vol. 25

October 2011

production/activity of COX-2 and tyrosine nitration of PGIS participate in increasing the substrate available for mPGES-1 activity and mPGES-1 expression, respectively. Tyrosine nitration of PGIS is most likely mediated by peroxynitrite (ONOO⫺) formed endogenously and generated in vivo by a rapid combination of NO and superoxide (O2䡠⫺). Although it is known that smoking increases oxidative stress and ROS production (13), the source of O2䡠⫺ involved in the tyrosine nitration of PGIS induced by cigarette smoking has not yet been identified. In particular, Mahfouz et al. (30) ruled out the possibility that the decreased production of PGI2 detected after incubation of cells with an aqueous extract of cigarette smoke depends on the activation of NADPH-oxidase. Our data add another key piece of information by showing that PGI2 modulates NADPHoxidase. In fact, blocking of the PGI2 pathway induces translocation of p47phox subunit from cytosol to plasma membrane and up-regulates ROS production. Supporting this concept, we observed that carbaprostacyclin, a stable analog of PGI2, prevents the activation of NADPH-oxidase induced by TS/IL-1␤. These data suggest that PGI2 plays a key role in the cardiovascular system not only by suppressing inflammation (33), inducing vasorelaxation, and inhibiting platelet and monocyte activation (34), but also by decreasing ROS production in specific pathological conditions, such as smoking. Thus, these observations confirm the data recently reported in neutrophils, endothelial, and/or smooth muscle cells exposed to phorbol ester (35), cytokines (36), or hypoxia (23). The importance of PGI2 in the modulation of different signal cascades regulated by cigarette smoke has been recently recognized. Increasing pulmonary production of PGI2 by lung-specific overexpression of PGIS suffices to reduce lung carcinogenesis (37) and to prevent pulmonary endothelial cell apoptosis induced by TS exposure (38). In this context, it is important to note that in our study, we have shown that in vascular endothelial cells, down-regulation of the PGI2/IP receptor pathway, mediated by silencing of PGIS, either by a PGI2 receptor antagonist, CAY10441, or by exposure to TS/IL-1␤, increases the synthesis and activation of mPGES-1. Since, in endothelial cells exposed to TS/IL-1␤, the increase of mPGES-1 expression is greater than that detected in samples treated with CAY10441, we can speculate that vascular mPGES-1 is not only regulated by PGI2, but also may be influenced by other transductional pathways. Finally, another important observation is that the PGI2-mediated activation of NADPH-oxidase up-regulates mPGES-1 expression and PGE2 production, suggesting that the availability of PGI2 is intimately involved in the molecular mechanisms underlying the concomitant higher expression of NADPH-oxidase and mPGES-1 detected in unstable plaques. In particular, we have shown that the expression of p47phox is up-regulated by the atherosclerotic process in vivo,


BARBIERI ET AL.

reflecting a potential critical role for vascular NADPHoxidase (39 – 41). An essential role for p47phox has been recently demonstrated by studies carried out in ApoE-knockout mice (39). In summary, our study shows that cigarette smoke in association with an inflammatory cytokine alters the balance between PGI2/PGE2, reducing the production of PGI2 and increasing the production of PGE2. The resulting prostanoid imbalance not only eliminates the vasodilatory, growth-inhibiting, antiplatelet aggregation, and antiadhesive effects of PGI2 (34), but also enhances PGE2-mediated augmentation of the activity of different genes, such as MMP-9 (9), angiopoietin (42), c-myc (43), and NADPH-oxidase (23), which favor vascular remodeling, angiogenesis, and cellular proliferation, pathophysiological events that, in turn, contribute to proinflammatory, prothrombotic, and proatherosclerotic consequences. The authors are grateful to Dr. A. Ieraci and Dr. D. Baldassarre for useful comments after reading this manuscript. This work was supported by Reti FIRB-2005RBPR05NWWC.

11.

12. 13.

14. 15.

16.

17.

18.

REFERENCES 1. 2.

3.

4.

5.

6. 7. 8.

9.

10.

Ambrose, J. A., and Barua, R. S. (2004) The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol. 43, 1731–1737 Barbieri, S. S., and Weksler, B. B. (2007) Tobacco smoke cooperates with interleukin-1beta to alter beta-catenin trafficking in vascular endothelium resulting in increased permeability and induction of cyclooxygenase-2 expression in vitro and in vivo. FASEB J. 21, 1831–1843 Knight-Lozano, C. A., Young, C. G., Burow, D. L., Hu, Z. Y., Uyeminami, D., Pinkerton, K. E., Ischiropoulos, H., and Ballinger, S. W. (2002) Cigarette smoke exposure and hypercholesterolemia increase mitochondrial damage in cardiovascular tissues. Circulation 105, 849 – 854 Lau, P. P., Li, L., Merched, A. J., Zhang, A. L., Ko, K. W., and Chan, L. (2006) Nicotine induces proinflammatory responses in macrophages and the aorta leading to acceleration of atherosclerosis in low-density lipoprotein receptor(-/-) mice. Arterioscler. Thromb. Vasc. Biol. 26, 143–149 Gross, N. D., Boyle, J. O., Morrow, J. D., Williams, M. K., Moskowitz, C. S., Subbaramaiah, K., Dannenberg, A. J., and Duffield-Lillico, A. J. (2005) Levels of prostaglandin E metabolite, the major urinary metabolite of prostaglandin E2, are increased in smokers. Clin. Cancer Res. 11, 6087– 6093 Reinders, J. H., Brinkman, H. J., van Mourik, J. A., and de Groot, P. G. (1986) Cigarette smoke impairs endothelial cell prostacyclin production. Arteriosclerosis 6, 15–23 Wang, M., Song, W. L., Cheng, Y., and Fitzgerald, G. A. (2008) Microsomal prostaglandin E synthase-1 inhibition in cardiovascular inflammatory disease. J. Intern. Med. 263, 500 –505 Cyrus, T., Ding, T., and Pratico, D. (2010) Expression of thromboxane synthase, prostacyclin synthase and thromboxane receptor in atherosclerotic lesions: correlation with plaque composition. Atherosclerosis 208, 376 –381 Cipollone, F., Fazia, M. L., Iezzi, A., Cuccurullo, C., De Cesare, D., Ucchino, S., Spigonardo, F., Marchetti, A., Buttitta, F., Paloscia, L., Mascellanti, M., Cuccurullo, F., and Mezzetti, A. (2005) Association between prostaglandin E receptor subtype EP4 overexpression and unstable phenotype in atherosclerotic plaques in human. Arterioscler. Thromb. Vasc. Biol. 25, 1925–1931 Wang, M., Zukas, A. M., Hui, Y., Ricciotti, E., Pure, E., and FitzGerald, G. A. (2006) Deletion of microsomal prostaglandin E synthase-1 augments prostacyclin and retards atherogenesis. Proc. Natl. Acad. Sci. U. S. A. 103, 14507–14512

SMOKE REGULATES mPGES-1 VIA PROSTACYCLIN/ROS PATHWAYS

19.

20.

21.

22. 23.

24.

25.

26.

27.

28.

Cheng, Y., Wang, M., Yu, Y., Lawson, J., Funk, C. D., and Fitzgerald, G. A. (2006) Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular function. J. Clin. Invest. 116, 1391–1399 Halliwell, B. (1989) Free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis. Br. J. Exp. Pathol. 70, 737–757 Csiszar, A., Podlutsky, A., Wolin, M. S., Losonczy, G., Pacher, P., and Ungvari, Z. (2009) Oxidative stress and accelerated vascular aging: implications for cigarette smoking. Front. Biosci. 14, 3128 –3144 Dworakowski, R., Alom-Ruiz, S. P., and Shah, A. M. (2008) NADPH oxidase-derived reactive oxygen species in the regulation of endothelial phenotype. Pharmacol. Rep. 60, 21–28 Cheng, S. E., Lee, I. T., Lin, C. C., Kou, Y. R., and Yang, C. M. (2010) Cigarette smoke particle-phase extract induces HO-1 expression in human tracheal smooth muscle cells: role of the c-Src/NADPH oxidase/MAPK/Nrf2 signaling pathway. Free Radic. Biol. Med. 48, 1410 –1422 Lin, C. C., Lee, I. T., Yang, Y. L., Lee, C. W., Kou, Y. R., and Yang, C. M. (2010) Induction of COX-2/PGE(2)/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation. Free Radic. Biol. Med. 48, 240 –254 Orosz, Z., Csiszar, A., Labinskyy, N., Smith, K., Kaminski, P. M., Ferdinandy, P., Wolin, M. S., Rivera, A., and Ungvari, Z. (2007) Cigarette smoke-induced proinflammatory alterations in the endothelial phenotype: role of NAD(P)H oxidase activation. Am. J. Physiol. Heart Circ. Physiol. 292, H130 –H139 Chung, K. F., Caramori, G., and Groneberg, D. A. (2004) Airway obstruction in chronic obstructive pulmonary disease. N. Engl. J. Med. 351, 1459 –1461 Barbieri, S. S., Ruggiero, L., Tremoli, E., and Weksler, B. B. (2008) Suppressing PTEN activity by tobacco smoke plus interleukin-1beta modulates dissociation of VE-cadherin/betacatenin complexes in endothelium. Arterioscler. Thromb. Vasc. Biol. 28, 732–738 Chattopadhyay, M., Velazquez, C. A., Pruski, A., Nia, K. V., Abdellatif, K. R., Keefer, L. K., and Kashfi, K. (2010) Comparison between 3-Nitrooxyphenyl acetylsalicylate (NO-ASA) and O2-(acetylsalicyloxymethyl)-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (NONO-ASA) as safe anti-inflammatory, analgesic, antipyretic, antioxidant prodrugs. J. Pharmacol. Exp. Ther. 335, 443–450 Barbieri, S. S., Cavalca, V., Eligini, S., Brambilla, M., Caiani, A., Tremoli, E., and Colli, S. (2004) Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms. Free Radic. Biol. Med. 37, 156 –165 Zou, M., Martin, C., and Ullrich, V. (1997) Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite. Biol. Chem. 378, 707–713 Chang, T. C., Huang, C. J., Tam, K., Chen, S. F., Tan, K. T., Tsai, M. S., Lin, T. N., and Shyue, S. K. (2005) Stabilization of hypoxia-inducible factor-1{alpha} by prostacyclin under prolonged hypoxia via reducing reactive oxygen species level in endothelial cells. J. Biol. Chem. 280, 36567–36574 Castro, P., Legora-Machado, A., Cardilo-Reis, L., Valenca, S., Porto, L. C., Walker, C., Zuany-Amorim, C., and Koatz, V. L. (2004) Inhibition of interleukin-1beta reduces mouse lung inflammation induced by exposure to cigarette smoke. Eur. J. Pharmacol. 498, 279 –286 Churg, A., Zhou, S., Wang, X., Wang, R., and Wright, J. L. (2009) The role of interleukin-1beta in murine cigarette smokeinduced emphysema and small airway remodeling. Am. J. Respir. Cell Mol. Biol. 40, 482– 490 Ekberg-Jansson, A., Andersson, B., Bake, B., Boijsen, M., Enanden, I., Rosengren, A., Skoogh, B. E., Tylen, U., Venge, P., and Lofdahl, C. G. (2001) Neutrophil-associated activation markers in healthy smokers relates to a fall in DL(CO) and to emphysematous changes on high resolution CT. Respir. Med. 95, 363–373 Pauwels, N. S., Bracke, K. R., Maes, T., Van Pottelberge, G. R., Garlanda, C., Mantovani, A., Joos, G. F., and Brusselle, G. G. (2010) Cigarette smoke induces PTX3 expression in pulmonary veins of mice in an IL-1 dependent manner. Respir. Res. 11, 134 Ryder, M. I., Saghizadeh, M., Ding, Y., Nguyen, N., and Soskolne, A. (2002) Effects of tobacco smoke on the secretion of

3739

29.

30.

31.

32.

33. 34. 35. 36.

3740

interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta from peripheral blood mononuclear cells. Oral Microbiol. Immunol. 17, 331–336 Oda, K., Tanaka, N., Arai, T., Araki, J., Song, Y., Zhang, L., Kuchiba, A., Hosoi, T., Shirasawa, T., Muramatsu, M., and Sawabe, M. (2007) Polymorphisms in pro- and anti-inflammatory cytokine genes and susceptibility to atherosclerosis: a pathological study of 1503 consecutive autopsy cases. Hum. Mol. Genet. 16, 592–599 Mahfouz, M., Qi, Z., and Kummerow, F. A. (2006) Inhibition of prostacyclin release by cigarette smoke extract in endothelial cells is not related to enhanced superoxide generation and NADPH-oxidase activation. J. Environ. Pathol. Toxicol. Oncol. 25, 585–595 Schmidt, P., Youhnovski, N., Daiber, A., Balan, A., Arsic, M., Bachschmid, M., Przybylski, M., and Ullrich, V. (2003) Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase. J. Biol. Chem. 278, 12813–12819 Zou, M. H., Daiber, A., Peterson, J. A., Shoun, H., and Ullrich, V. (2000) Rapid reactions of peroxynitrite with heme-thiolate proteins as the basis for protection of prostacyclin synthase from inactivation by nitration. Arch. Biochem. Biophys. 376, 149 –155 Moncada, S., and Vane, J. R. (1981) Prostacyclin and the vascular endothelium. Bull. Eur. Physiopathol. Respir. 17, 687–701 Wu, K. K., and Thiagarajan, P. (1996) Role of endothelium in thrombosis and hemostasis. Annu. Rev. Med. 47, 315–331 Umeki, S. (1994) Prostaglandin E and analogs of prostacyclin influencing superoxide production by the human neutrophil NADPH oxidase system. Int. J. Biochem. 26, 1003–1008 Muzaffar, S., Shukla, N., Angelini, G., and Jeremy, J. Y. (2004) Nitroaspirins and morpholinosydnonimine but not aspirin inhibit the formation of superoxide and the expression of gp91phox induced by endotoxin and cytokines in pig pulmonary artery vascular smooth muscle cells and endothelial cells. Circulation 110, 1140 –1147

Vol. 25

October 2011

37.

38.

39.

40.

41.

42. 43.

Keith, R. L., Miller, Y. E., Hudish, T. M., Girod, C. E., SottoSantiago, S., Franklin, W. A., Nemenoff, R. A., March, T. H., Nana-Sinkam, S. P., and Geraci, M. W. (2004) Pulmonary prostacyclin synthase overexpression chemoprevents tobacco smoke lung carcinogenesis in mice. Cancer Res. 64, 5897–5904 Nana-Sinkam, S. P., Lee, J. D., Sotto-Santiago, S., Stearman, R. S., Keith, R. L., Choudhury, Q., Cool, C., Parr, J., Moore, M. D., Bull, T. M., Voelkel, N. F., and Geraci, M. W. (2007) Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke. Am. J. Respir. Crit. Care Med. 175, 676 – 685 Barry-Lane, P. A., Patterson, C., van der Merwe, M., Hu, Z., Holland, S. M., Yeh, E. T., and Runge, M. S. (2001) p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice. J. Clin. Invest. 108, 1513–1522 Niu, X. L., Madamanchi, N. R., Vendrov, A. E., Tchivilev, I., Rojas, M., Madamanchi, C., Brandes, R. P., Krause, K. H., Humphries, J., Smith, A., Burnand, K. G., and Runge, M. S. (2010) Nox activator 1: a potential target for modulation of vascular reactive oxygen species in atherosclerotic arteries. Circulation 121, 549 –559 Vendrov, A. E., Hakim, Z. S., Madamanchi, N. R., Rojas, M., Madamanchi, C., and Runge, M. S. (2007) Atherosclerosis is attenuated by limiting superoxide generation in both macrophages and vessel wall cells. Arterioscler. Thromb. Vasc. Biol. 27, 2714 –2721 Pichiule, P., Chavez, J. C., and LaManna, J. C. (2004) Hypoxic regulation of angiopoietin-2 expression in endothelial cells. J. Biol. Chem. 279, 12171–12180 Shin, V. Y., Liu, E. S., Ye, Y. N., Koo, M. W., Chu, K. M., and Cho, C. H. (2004) A mechanistic study of cigarette smoke and cyclooxygenase-2 on proliferation of gastric cancer cells. Toxicol. Appl. Pharmacol. 195, 103–112


Received for publication March 17, 2011. Accepted for publication July 1, 2011.

BARBIERI ET AL.